Hepatitis A virus (HAV) is a morphologically, biochemically, and immunologically distinct picornavirus that is the etiological agent of infectious hepatitis in humans. Like other picornaviruses, HAV contains a single-stranded, positive-sense, infectious RNA genome. Four major capsid proteins have been described, VP1 (32-33 Kilodaltons or KDa), VP2 (26-29 KDa), VP3 (22-27 KDa) and VP4 (10-14 KDa). HAV has now been cloned and sequenced, e.g., Najarian, R. et al. Proc. Natl. Acad. Sci. 82, 2627 (1985). For recent reviews, see, for example, Gerety, R. J. (ed.) Hepatitis A Academic Press 1984; Feinstone, S. M. Progress in Liver Diseases, 8, 299 (1986); and Mijch, A. M. et al. Seminars in Liver Diseases 6, 42 (1986).
Various methods have been worked out to partially purify HAV virions for the purposes of study and initial characterization. See, for example, Hornbeck, C. L. et al., Interviroloy 6; 309-314 (1975); Locarnini, S. A. et al., Intervirology 10; 300-308 (1978); Siegl, G. et al., J. Virol. 26, 40-47 (1978); Siegl, G. et al., J. Gen. Virol. 57, 331-341 (1981); Siegl, G. et al., Intervirology 22, 218 (1984); Hughes, J. V. et al., J. Virol. 52, 465 (1984); and Wheeler, C. M. et al., J. Virol, 58, 307 (1986). Each of these methods employs one or more steps that are likely to prevent approval by the Food and Drug Administration for the purposes of testing and then selling a safe vaccine against HAV. For example, detergents and/or exogenous enzymes are commonly used. Furthermore, all of these methods employ unwieldy, impractical and excessively expensive steps, such as sucrose gradient centrifugation or CsCl-density gradient centrifugation.
Applicants have discovered methods of obtaining very pure HAV without the use of detergents or exogenous enzymes. Furthermore, the methods disclosed herein can be readily scaled up to commercial production. In addition, applicants have succeeded in adapting these methods to MRC-5 host cells, which are certified by the Food and Drug Administration for human vaccine production.